Muscarinic acetylcholine receptors modulate the function of a variety of effectors through heterotrimeric G proteins. A prenylated peptide specific to the G protein γ5 subunit type inhibits G protein activation by the M2 muscarinic receptor in a reconstitution assay. Scrambling the amino acid sequence of the peptide significantly reduces the efficacy of the peptide. The peptide does not disrupt the G protein heterotrimer. In cultured sympathetic neurons, the γ5 peptide inhibits modulation of Ca2+ current by the M4 receptor. Peptide activity is specific, the scrambled peptide and peptides specific to two other members of the G protein γ subunit family are significantly less effective. The γ5 peptide has no effect on Ca2+ current modulation by the α2-adrenergic and somatostatin receptors. In addition, the γ5 peptide inhibits muscarinic receptor signaling in spinal cord slices with specificity. These results support a specific role for G protein γ subunit types in signal transduction, most likely at the receptor-G protein interface.